Laboratory Issues

The most important laboratory issue of concern, particularly with respect to PCR analysis, is the avoidance of contamination. When performing PCR for EBV or KSHV repeatedly, it is essential to use extreme care in separating the PCR products from the reagents and specimens. When using ultrasensitive techniques, such as nested PCR, the potential for contamination is intensified. Complete separation of pre- and post-PCR steps should be maintained. Cross contamination among patient samples and controls should be avoided. PCR primers for specific assays tend to differ among various laboratories, such that each laboratory validates and sets normal ranges for the assay.

An issue that is specific to AIDS-related lymphomas is the concern of working with HIV-infected tissues. While the lymphoma cells do not carry the HIV genome, the tissue may have infected T cells or macrophages. If DNA is being extracted from formalin-fixed, paraffin-embedded tissues, infectious HIV is not a concern, as any viral particles would have been inactivated. However, when extracting nucleic acids from fresh and frozen tissues, extreme care should be taken and the individual doing the extraction should be informed of the risks and be properly trained. Nucleic acid extraction from fresh or frozen tissue should be done at a BSL-2 biosafety level, which essentially requires the procedure to be done in a laminar flow hood and for the technologist to wear gloves and a laboratory coat. Since blood-borne pathogen exposure is a risk when working with any tissue, and HIV status is frequently unknown, the use of gloves and a lab coat are required for every extraction involving fresh or frozen tissues (universal precautions).

A specific difficulty for performing some of the onco-gene and virus detection assays is obtaining good positive controls. For MYC translocations, any Burkitt lymphoma cell line can be used, and many are available from the American Type Culture Collection (ATCC). For BCL6 mutations, controls are more difficult to obtain, as SSCP involves amplification of separate but overlapping regions, with different controls required for each region. Thus, controls will vary with the amplification fragment established by the individual laboratory. Several diffuse large B-cell lymphoma lines have BCL6 mutations, or patient specimens with BCL6 mutations can be used as controls. For virus detection, several EBV-positive lymphomas and lym-phoblastoid cell lines, as well as KSHV-positive primary effusion lymphomas, are available from the ATCC and the AIDS and Cancer Specimen Bank (ACSB) repositories. PEL

cell lines are available through ATCC that contain both EBV and KSHV, which can be used as a positive control for multiple assays.

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